CN104673829A - Novel application of beta-expansion protein gene GmEXPB2 - Google Patents
Novel application of beta-expansion protein gene GmEXPB2 Download PDFInfo
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Abstract
The invention discloses a novel application of beta-expansion protein gene GmEXPB2, and particularly discloses a novel application of beta-expansion protein gene GmEXPB2 in improvement of nitrogen use efficiency of leguminous plants. The inventor of the invention identifies the beta-expansion protein gene GmEXPB2 highly expressed in the early stage formed in soybean root nodules through a quantitative PCR and chemical tissue positioning method, and the gene participates in the control of development of nodule primodium, cortical cells, parenchyma cells and early nodule microtubule organization. The research on the transgenic composite plants and intact transformed plants of soybean shows that over expression of GmEZPB2 remarkably increases the quantity and weight of soybean root nodules and the nitrogen content of the plants and finally increases the biomass of the transgenic plants, and the research of the inventor provides a gene source for nitrogen efficiency and molecular breeding of leguminous plants including soybean and has theoretical and practical significance to development of environment-friendly sustainable agriculture.
Description
Technical field
The present invention relates to the new opplication of gene, being specifically related to β-expansin gene GmEXPB2 improving the new opplication in leguminous plants nitrogen efficiency, belonging to biological technical field.
Background technology
Soybean [Glycine max (L.) Merr] originates from China, is grain important in world wide, oil plant, feed and energy crop, occupies critical role (Palander et al., 2005) in people's life food configuration.In recent years, along with the growth of soybean consumption and the develop rapidly of aquaculture, there is serious unbalance in domestic soybean supply and demand, the contradiction between soybean in China and demand.Within 2000, rise, China just becomes importer (Yang Wenyu etc. 2008) maximum in the world now from traditional soybean export state.Latest data shows, within 2013, China's soybean import reaches 6,338 ten thousand tons, increase by 8.6% on year-on-year basis, the high position that sets a record (Chinese grain information net, http://www.chinagrain.cn/dadou/2014/1/13/201411313313124408.sht ml).Therefore, for meeting the domestic needs to soybean consumption, China is necessary to greatly develop Soybean Industry, solves supply and demand of soybean contradiction, to reduce the dependence to Soybean import.
Soybean in China level is lower, and mainly soil nutrient availability of tracing it to its cause is lower, such as nitrogen starved plot, is one of principal element of restriction Soybean production.In long-term evolutionary process, plant defines the mechanism of action of a series of adaptation Low nitrogen stress.Wherein, nodulation and nitrogen fixation is the important channel of improving leguminous crop nitrogen efficiency.Root nodule bacterium infect the syntaxial system that leguminous plants root system is set up, significant to the Sustainable development of agriculture production and ecotope.Research shows, Rhizobium Inoculation can improve the nitrogen fixing capacity of leguminous crop, improves nitrogen nutrition, promotes root growth and improves crop yield (Ferreira et al., 2009).In the soybean main product state such as the U.S., Brazil, Rhizobium Inoculation is one of major measure becoming soybean yield-increasing, spread and application.And legume inoculation technology is applied to agriculture production by China, also achieve good benefit (Tang Fuyue etc. 2011; Mendes et al., 2003; Sogut 2006; De Freitas et al., 2012).
The formation of root nodule and organ expand with the morphogenesis of cell walls closely related, and expansin (Expansin) is a class cell wall expansion protein, increase and play an important role in softening, Nodule Growth growth etc. at inducing cell wall.
But, very limited about the research of expansin in regulation and control leguminous crop nodulation and nitrogen fixation, and in soybean, there is not been reported for the expansin gene of regulation and control nodule formation and growth.
Summary of the invention
Based on above-mentioned research background, contriver organizes localization method by quantitative PCR and chemistry, identifies one and forms the early stage β-expansin gene GmEXPB2 highly expressed at soybean nodulation.This gene has participated in the growth of the former base of root nodule, tegumental cell, parenchyma cell and early stage root nodule microtubule tissue.
Utilize soybean transgene compound plant and the research of whole strain transformed plant to show, overexpression GmEXPB2 significantly increases soybean nodulation quantity and weight, improves N content of crop tissue, ultimately increases the biomass of transfer-gen plant.
Efficient and high yield molecular breeding provides genetic resources by the leguminous crop nitrogen for comprising soybean for the research of contriver.
Usefulness of the present invention is: this research to increase comprise the leguminous crop root nodule numbers of soybean and weight, raising N content of crop tissue, increase transfer-gen plant biomass significant, to development environment friendly sustainable agriculture, also there is important practice significance, simultaneously also for high yield molecular breeding provides genetic resources.
Accompanying drawing explanation
Fig. 1 is GmEXPB2 Gene Expression Profile Analysis;
Fig. 2 is the dissection and analysis of GmEXPB2 promoters driven GUS expressive site in soybean transgene compound plant;
Fig. 3 be excessive, interfere GmEXPB2 on the impact of soybean transgene compound plant dross;
Fig. 4 be excessive, interfere GmEXPB2 impact on soybean transgene compound plant strain growth and nitrogen/phosphorus content under the low-phosphorous process of height;
Fig. 5 is the impact of overexpression GmEXPB2 on Soybean transgenic plant Root morphology;
Fig. 6 is the infection processs of the genetically engineered soybean Rhizobium Inoculation USDA110-GFP of overexpression GmEXPB2;
Fig. 7 be under high and low phosphorus process overexpression GmEXPB2 on the impact of soybean nodulation;
Fig. 8 is overexpression GmEXPB2 on the impact of soybeans they grow and nitrogen/phosphorus content.
Embodiment
Below in conjunction with the drawings and specific embodiments, concrete introduction is done to the present invention.
One, β-expansin gene GmEXPB2 expression pattern analysis
In the soybean gene group database announced, by homology comparison, dope soybean β-expansin family and have 9 members, by quantitative PCR technique, determine that GmEXPB2 gene is the gene of root nodule Enhanced expressing.The open reading frame length of GmEXPB2 gene is 834bp, and 278 amino acid whose albumen of encoding, belong to β class cell walls expansin.Existing research shows, this gene and nodulation and nitrogen fixation closely related, first analyze its expression pattern.
Soybean seeds HN66 3% (v/v) H
2o
2surface sterilization one minute, Germination 5 days in the quartz sand that 1/2 pancebrin is wetting, Rhizobium Inoculation transfers to low nitrogen (nitrogen: 530 μMs of NH after 1 hour
4nO
3+ KNO
3+ Ca (NO
3)
24H
2o) process in nutritive medium.Gather in the crops the process root nodule of the 7th day respectively, the root of the 18th day, stem, leaf, flower and the process beanpod of the 29th day and seed extracting RNA.For the expression amount of comparative analysis GmEXPB2 in different growing stage root nodule, the 7th day root system of the root system of the 4th day, the root nodule of the 7th, 14,21,30,40 day and non-seeded root nodule bacterium after results Rhizobium Inoculation, and extract the RNA of corresponding site, reverse transcription becomes cDNA, uses the expression pattern of quantitative PCR detection GmEXPB2 further.The house-keeping gene EF-1a of soybean is as internal reference.Primer for quantitative PCR detection gene expression amount is respectively:
The primer of soybean EF-1a gene is:
EF-1a F:5’-TGCAAAGGAGGCTGCTAACT-3’(SEQ ID NO:1)
EF-1a R:5’-CAGCATCACCGTTCTTCAAA-3’(SEQ ID NO:2)
The primer of GmEXPB2 gene is:
GmEXPB2F:5’-TGGTGCTTGTGGTTATGGAAGT-3’(SEQ ID NO:3)
GmEXPB2R:5’-TGAACCACACCCAGGACAGCT-3’(SEQ ID NO:4)
Quantitative PCR response procedures is: RNA sample reverse transcription gained cDNA is diluted 50 times as quantitative PCR reaction template.Choose appropriate cDNA stoste and do the template that gradient dilution is typical curve.Adopt 20 μ L reaction systems in test, comprising: 2 × SYBR Green PCR master mix of 10 μ L, 10 μMs of forward and reverse primers of each 0.6 μ L, the cDNA that 2 μ L dilute, finally mend to 20 μ L with Mili-Q water.
Quantitative PCR reaction conditions is: 95 DEG C of sex change 1 minute, then 94 DEG C of cracking 15 seconds, 60 DEG C in conjunction with 15 seconds, 72 DEG C extend 30 seconds and carry out 40 circulations.
The expression amount of each sample is calculated with the Real-Time Analysis Software 6.0 of Rotor-Gene.
Fig. 1 is GmEXPB2 Gene Expression Profile Analysis.Wherein:
Fig. 1 (A) is the expression of GmEXPB2 in different tissues;
Fig. 1 (B) is the expression of GmEXPB2 in different development stage root nodule.
In figure, data are mean value and standard errors of 3 biology repetitions.
Result shows:
(1) expression amount of GmEXPB2 in root nodule is the highest, and root takes second place, in beanpod, also there is trace expression, but can't detect the accumulation of this gene mRNA in stem, leaf, flower and seed, see Fig. 1 (A).
(2) qRT-PCR is carried out to the root nodule of different development stage and detect analysis, find GmEXPB2 Enhanced expressing in the root system of inoculation after 4 days, when Nodule Growth reaches maximum value to expression amount when the 7th day, but the prolongation of the growth time along with root nodule, its expression amount reduces gradually, when Nodule Growth was by 30 and 40 days, almost can't detect the expression of this gene, see Fig. 1 (B), hint GmEXPB2 participates in the growth course of the early stage root nodule of regulation and control.
Two, GmEXPB2 gene promoter clone, vector construction and tissue expression positioning analysis
Conventionally, extract soybean leaves or root genomic dna, take Soybean genomic DNA as template, by upstream specific primer and downstream specific primers amplify GmEXPB2 promotor 2742bp fragment, after PCR fragment recovery order-checking is errorless, after double digestion being carried out to recovery fragment and object carrier by PstI and NcoI, GmEXPB2 gene is connected to object carrier pCAMBIA3301.Wherein,
Upstream specific primer is:
5’-
CTGCAGACCTTCCTGACTTCCCGAAT-3’(SEQ ID NO:5)
Downstream special primer is:
5’-
CCATGGGTGTAGGAGCCATAATATCACAACC-3’(SEQ ID NO:6)
After obtaining transgenic hairy root by agriculture bacillus mediated soybean hypocotyl injection conversion method, main root is cut, retain from callus director hairly root out, be immersed in by hairly root in root nodule bacterium bacterium liquid and move into water planting CMC model after 1 hour, mill water culture nutrient solution is above-mentioned low nitrogen process.For determining the tissue positioned of GmEXPB2 in soybean nodulation different developmental phases, gather in the crops the Rhizobium Inoculation process root nodule of 4,7,14,21 and 30 days respectively and carry out GUS staining analysis, the tissue positioned of genetic expression can be observed further by paraffin section, the results are shown in Figure 2.
See Fig. 2, the dissection and analysis of GmEXPB2 promoters driven GUS expressive site in soybean transgene compound plant, wherein:
Fig. 2 (A)-Fig. 2 (H) and Fig. 2 (L) is the transgenosis root nodule of expressing GmEXPB2 promoters driven GUS;
The profile that Fig. 2 (A)-Fig. 2 (D) is soybean nodulation;
Fig. 2 (E)-Fig. 2 (H) and Fig. 2 (L) is soybean nodulation crosscut;
Fig. 2 (I)-Fig. 2 (K) is for expressing CaMV35S contrast;
Fig. 2 (I) is soybean nodulation rip cutting;
The crosscut that Fig. 2 (J) and Fig. 2 (K) is soybean nodulation.
Soybean transgene compound plant strain growth containing in Poor nitrogen nutrition liquid, wherein:
Fig. 2 (A) and Fig. 2 (E) is growth 4 days root nodules;
Fig. 2 (B) and Fig. 2 (F) is 7 days root nodules;
Fig. 2 (C), Fig. 2 (G), Fig. 2 (I), Fig. 2 (J) are 14 days root nodules;
Fig. 2 (L) is 21 days root nodules;
Fig. 2 (D) and Fig. 2 (H) is growth 30 days root nodules.
NVT is that early stage root nodule does up the beginning; NVB is root nodule vascular bundle; Pa represents parenchyma cell; EN represents tegumental cell.
The scale of Fig. 2 (A) is 20 μm, and the scale of Fig. 2 (B), Fig. 2 (C), Fig. 2 (E) and Fig. 2 (F) is 50 μm, and in all the other figure, scale is 100 μm.
Result shows: compared with the contrast of constitutive expression [Fig. 2 (I), Fig. 2 (J) and Fig. 2 (K)], Rhizobium Inoculation is after 4 days, and the soybean root system center pillar of GmEXPB2 mainly corresponding to root nodule happening part and root nodule former base initial part bit table reach [Fig. 2 (A) and Fig. 2 (E)]; When growing 7 days, in Nodule cortex and parenchyma cell that the vascular tissue and growing that GUS staining signals is mainly present in root and root nodule connecting portion is formed [Fig. 2 (B) and Fig. 2 (F)]; Grow in the root nodule of 14 days, the vascular tissue that root nodule and root system intersect presents two bifurcation state, and GmEXPB2 can along with the differentiation of Nodule cortex and parenchyma cell and Enhanced expressing [Fig. 2 (C) and Fig. 2 (G)]; When Nodule Growth was by 21 days, GUS dyeing disappears in root nodule vascular tissue, mainly in cortex and parenchyma [Fig. 2 (L)]; GUS staining signals [Fig. 2 (D) and Fig. 2 (H)] is almost can't detect in the section of the growth root nodule of 30 days.
Three, excessive, interfere GmEXPB2 on the impact of soybean transgene compound plant dross
That the soybean hypocotyl injection that utilizes Agrobacterium rhyzogenesK599 to mediate obtains is excessive, interfere GmEXPB2 transgenosis compound plant (root be transgenic hairy root, overground part be non-transgenic).Can main root be cut when wherein transgenic hairy root growth length is about 10cm, remove the adventive root grown at non-wound.Follow-up phenotypic evaluation all uses this strain, and CK is contrast.
After hair root grows side root, root samples is taked to extract RNA, transgenosis hair root selection markers is Totomycin, and available hygromycin gene (Hyg) detects the true and false of hair root and retains a transgenosis root system carries out Rhizobium Inoculation and high phosphorus (500 μMs of KH
2pO
4), low-phosphorous (10 μMs of KH
2pO
4) process sand culture test.The results growth root nodule of 7 days, extract RNA, use the effect of quantitative PCR detection overexpression and interference further, in quantitative PCR assays with soybean house-keeping gene with EF-1a described above for reference gene, the expression amount of gene GmEXPB2 and the ratio of house-keeping gene expression amount for the purpose of relative expression quantity.
Quantitative PCR step:
(1) primer of Hyg gene is:
Hyg F:5’-GCTGTTATGCGGCCATTGTC-3’(SEQ ID NO:7)
Hyg R:5’-GACGTCTGTCGAGAAGTTTC-3’(SEQ ID NO:8)
The primer of soybean EF-1a gene is the same;
The primer of GmEXPB2 gene is the same.
(2) reaction system:
2×SYBR Green PCR master mix：10μL
Upstream primer. (10 μm of ol/L): 0.6 μ L
Downstream primer. (10 μm of ol/L): 0.6 μ L
Mili-Q water: mend to 20 μ L
The cDNA:2 μ L of dilution
(3) reaction conditions:
Confirm to obtain effective different transgenic line through the detection of resistant gene and quantitative PCR, see Fig. 3 (A) and Fig. 3 (B).
The different transgenic lines of results Rhizobium Inoculation after 50 days measure relative physiologic index, comprising: root nodule dry weight, root nodule number, root nodule size etc., wherein root nodule size is the dry weight (ratio of root nodule dry weight and root nodule number) of single root nodule.Biomass: after the root nodule of root is taken counting, take fresh weight with ten thousand/balance, all samples completes at 105 DEG C of baking ovens and within 30 minutes, is placed on 75 DEG C of oven dry to constant weight, takes dry weight.
Fig. 3 be excessive, interfere GmEXPB2 on the impact of soybean transgene compound plant dross.Wherein:
Fig. 3 (A) is PCR detection positive plant;
Fig. 3 (B) is quantitative PCR detection GmEXPB2 expression amount;
Fig. 3 (C) is root nodule picture;
Fig. 3 (D) is root nodule number;
Fig. 3 (E) is root nodule dry weight;
Fig. 3 (F) is root nodule size.
Sample tentatively detects positive plant by qualitative PCR, and EF-1a (gene order number: X56856) is soybean house-keeping gene, and Hyg is carrier target gene.The expression amount of gene GmEXPB2 and the ratio of EF-1a expression amount for the purpose of relative expression quantity.
CK is contrast strain; OX is the overexpression strain of GmEXPB2; RNAi is the interference strain of GmEXPB2.Be mean value and the standard error of 4 the biology repetitions of same transgenic plant in figure.
Asterisk represents that same proterties is in OX or RNAi strain and the significant difference contrasted between CK strain:
When * representing conspicuous level 0.01< ρ≤0.05, significant difference;
When * represents conspicuous level 0.001< ρ≤0.01, the significance of difference is between significantly and extremely significantly;
When * * represents conspicuous level ρ≤0.001, difference is extremely remarkable;
Ns represents that difference is not remarkable.
Result shows: compared with the control, overexpression GmEXPB2 (OX) significantly promotes the growth of root nodule, add root nodule number and dry weight, GmEXPB2 (RNAi) is interfered then to significantly suppress the growth of root nodule, decrease root nodule number, reduce root nodule weight and reduce root nodule size, the formation of root nodule of GmEXPB2 major effect is described.
Four, excessive, interfere GmEXPB2 impact on soybean transgene compound plant strain growth and nitrogen/phosphorus content under the low-phosphorous process of height
Biomass measures: one of percentage balance weighs overground part and root samples fresh weight, and all samples completes at 105 DEG C of baking ovens and within 30 minutes, is placed on 75 DEG C of oven dry to constant weight, takes dry weight.
Plant nitrogen, phosphorus measure: (model is SAN++ for plant nitrogen and phosphorus content employing Continuous Flow Analysis instrument, originate from Holland) measure, first take Plant samples about 0.2g, add the 5mL vitriol oil and clear up rear distilled water constant volume and cause 50mL, and diluted sample 4 is doubly made liquid to be measured.For the mensuration of plant ammonia nitrogen, using Sodium Nitroprusside as catalyzer, make the ammonium nitrogen of testing sample and sodium salicylate and sodium hypochlorite reaction generate blue compound, at 660nm wavelength, place measures its absorption value; For the mensuration of plant phosphorus content, under antimonic salt exists, the phosphato-molybdic heteropolyacid that ortho-phosphoric acid and ammonium molybdate reaction generate, P-Mo blue is generated by xitix reduction after heating bath, flow into detector again in 880nm wavelength place colorimetric, finally colorimetric signal is inputted computer, and by Flow Access computed in software result.
Nutrient content in plant unit plant nitrogen, phosphorus amount represent, calculation formula is:
Nitrogen content (mg/plant)=nitrogen concentration (mg/g) × plant weights (g/plant)
Phosphorus content (mg/plant)=phosphorus concentration (mg/g) × plant weights (g/plant)
Fig. 4 be excessive, interfere GmEXPB2 impact on soybean transgene compound plant strain growth and nitrogen/phosphorus content under the low-phosphorous process of height.Wherein:
Fig. 4 (A) is the phenotype analytical of high low-phosphorous process;
Fig. 4 (B) is plant weights;
Fig. 4 (C) is nitrogen content;
Fig. 4 (D) is phosphorus content.
CK is empty vector control; OX is overexpression GmEXPB2 transgenosis compound plant; RNAi is for interfering GmEXPB2 transgenosis compound plant.
Testing data is mean value and the standard error of four biology repetitions.
Asterisk represent the dry weight of GmEXPB2 gene, nitrogen and phosphorus content OX and RNAi strain with contrast otherness between strain and compare (t-checks):
When * representing conspicuous level 0.01< ρ≤0.05, significant difference;
When * represents conspicuous level 0.001< ρ≤0.01, the significance of difference is between significantly and extremely significantly.
Result shows: overexpression GmEXPB2 significantly promotes the growth of dross soybean, improves nitrogen/phosphorus efficiency and the biomass of soybean transgene compound plant, and interferes GmEXPB2 to significantly suppress soybeans they grow.
Five, overexpression GmEXPB2 is on the impact of Soybean transgenic plant Root morphology
The transgenosis of the overexpression GmEXPB2 of full uniformity and Wild-type soy are invaded bubble to showing money or valuables one carries unintentionally in 1/4 soybean (Kidney bean) pancebrin, the filter paper that nutritive medium is soaked in utilization carries out vernalization growth.Measure the growth main root root-hair zone of 2 days and non-root-hair zone length under Stereo microscope respectively, and compare the growth Soybean Root Hairs density of 2 days further, each process has ten biology to repeat.
Fig. 5 is the impact of overexpression GmEXPB2 on Soybean transgenic plant Root morphology.Wherein:
Fig. 5 (A) is soybean phenotype;
Fig. 5 (B) is root hair phenotype;
Fig. 5 (C) is main root length.
OX1-3 is the overexpression strain of GmEXPB2; WT1-2 is wild-type.
Be the data that same transgenic plant 10 are repeated in figure.
Asterisk represents the significant difference of same proterties between OX and WT strain: when * * * represents conspicuous level ρ≤0.001, difference is extremely remarkable.
Result shows: GmEXPB2 is to the important regulating and controlling effect that grows of soybean main root and root hair, and data statistics finds, overexpression GmEXPB2 significantly promotes the growth of soybean root system, adds that main root is long, root gross density and length.
Six, overexpression GmEXPB2 impact that genetically engineered soybean is infected
First coil paper vernalization overexpression GmEXPB2 and Wild-type soy seed, grow the root nodule bacterium USDA110 (USDA110-GFP after 4 days, soybean seedling root system being immersed GFP mark, teach laboratory by Tan Zhiyuan to be so kind as to give) in bacterium liquid about 1 hour, again plant is transferred in sand culture and cultivate, and under Laser Scanning Confocal Microscope, observe the developmental state infecting line and the former base of root nodule.
Fig. 6 is the infection processs of the genetically engineered soybean Rhizobium Inoculation USDA110-GFP of overexpression GmEXPB2, wherein:
Fig. 6 (A)-Fig. 6 (F) for inoculation after 2 days root nodule bacterium stick to territory, root-hair zone;
Fig. 6 (G)-Fig. 6 (L) infects line (IT) for inoculation and is formed after 3 days;
Fig. 6 (M)-Fig. 6 (R) for inoculation after 4 days IT continued growth to root tegumental cell.
Fig. 6 (A)-Fig. 6 (C), Fig. 6 (G)-Fig. 6 (I), Fig. 6 (M)-Fig. 6 (O) are wild-type; Fig. 6 (D)-Fig. 6 (F), Fig. 6 (J)-Fig. 6 (L), Fig. 6 (P)-Fig. 6 (R) are the genetically engineered soybean of overexpression GmEXPB2.
The scale of Fig. 6 (A)-Fig. 6 (F) is 100 μm, and the scale of other pictures is 50 μm.
Result shows: GmEXPB2 by lax cell walls, can increase the adhered area of root nodule bacterium, and then promotes the formation infecting and accelerate root nodule of root nodule bacterium.
Seven, overexpression GmEXPB2 is on the impact of genetically engineered soybean dross
Overexpression GmEXPB2 is tested the functional analysis of soybean nodulation, the transgenosis of GmEXPB2 overexpression and Wild-type soy seed (OX1-3: the transgenic line of overexpression GmEXPB2; WT: wild-type), surface sterilization also to be sprouted in sterilized sand 5 days, then seedling inoculation rihizobium japonicum BXYD3 consistent for growing way is moved into hydroponic system after mono-hour.Plant strain growth in above-mentioned Poor nitrogen nutrition liquid and carry out high phosphorus (500 μMs) and low-phosphorous (5 μMs) process.Change weekly one time of nutrition liquid and keep pH to be about 5.8.The results inoculation soybean nodulation of latter 25 days carries out root nodule number, dry weight and nitrogenase activity determination analysis.
Fig. 7 be under high low-phosphorous process overexpression GmEXPB2 on the impact of soybean nodulation.Wherein:
Fig. 7 (A) is root nodule picture;
Fig. 7 (B) is root nodule number;
Fig. 7 (C) is root nodule dry weight;
Fig. 7 (D) is nodule nitrogenase activities.
Transgenosis and Wild-type soy are at different phosphate concentration (high phosphorus: 500 μMs of KH
2pO
4; Low-phosphorous: 10 μMs of KH
2pO
4) Poor nitrogen nutrition liquid in growth 25 days.WT is wild-type; OX1-3 is GmEXPB2 overexpression strain.Be mean value and the standard error of 4 the biology repetitions of same transgenic plant in figure.
Asterisk represents the significant difference of same proterties between OX and WT strain:
When * representing conspicuous level 0.01< ρ≤0.05, significant difference;
When * represents conspicuous level 0.001< ρ≤0.01, the significance of difference is between significantly and extremely significantly;
When * * represents conspicuous level ρ≤0.001, difference is extremely remarkable;
Ns represents that difference is not remarkable.
Result shows: overexpression GmEXPB2 significantly promotes soybean nodulation, adds the nodule nitrogenase activities under the root nodule numbers under high and low phosphorus condition, root nodule dry weight and high phosphorus condition.
Eight, overexpression GmEXPB2 is on the impact of soybeans they grow and nitrogen/phosphorus content
Biomass and nitrogen, phosphorus determination method are the same.
Fig. 8 is overexpression GmEXPB2 on the impact of soybeans they grow and nitrogen/phosphorus content.Wherein,
Fig. 8 (A) is plant phenotype;
Fig. 8 (B) is plant weights;
Fig. 8 (C) is nitrogen content;
Fig. 8 (D) is phosphorus content.
The transgenosis of Rhizobium Inoculation process and Wild-type soy are at different phosphate concentration (high phosphorus: 500 μMs of KH
2pO
4; Low-phosphorous: 10 μMs of KH
2pO
4) the process of Poor nitrogen nutrition liquid under growth 25 days.WT is wild-type; OX1-3 is GmEXPB2 overexpression strain.Be mean value and the standard error of 4 the biology repetitions of same transgenic plant in figure.
Asterisk represents the significant difference of same proterties between OX and WT strain:
When * representing conspicuous level 0.01< ρ≤0.05, significant difference;
When * represents conspicuous level 0.001< ρ≤0.01, the significance of difference is between significantly and extremely significantly;
When * * represents conspicuous level ρ≤0.001, difference is extremely remarkable;
Ns represents that difference is not remarkable.
Result shows: overexpression GmEXPB2 by promoting soybean nodulation fixed nitrogen, and then facilitates soybeans they grow, under high and low phosphorus condition, significantly improves the content of nitrogen and phosphorous and the biomass of plant.
In sum, β-expansin gene GmEXPB2 has the New function of regulating and controlling soybean nodulation and nitrogen fixation.Under low nitrogen process Rhizobium Inoculation condition, the root nodule numbers of the overexpression soybean transgene compound plant of gene of the present invention, weight, nitrogen/phosphorus content and biomass are all significantly higher than contrast strain, GmEXPB2 cultivating efficient nodulation and nitrogen fixation transgenic leguminous plants, improve the fertilizer utilization efficiency of farm crop and improve in crop yield there is positive application value.
It should be noted that, above-described embodiment does not limit the present invention in any form, the technical scheme that the mode that all employings are equal to replacement or equivalent transformation obtains, and all drops in protection scope of the present invention.
Claims (6)
1. β-expansin gene GmEXPB2 is improving the new opplication in leguminous plants nitrogen efficiency.
2. new opplication according to claim 1, is characterized in that, described β-expansin gene GmEXPB2 grows early expression at legume nodule, and is positioned the former base of root nodule, originally microtubule tissue, Nodule cortex, parenchyma cell and early stage vascular bundle.
3. new opplication according to claim 1, it is characterized in that, compared with Wild-type soy, the genetically engineered soybean of overexpression GmEXPB2 significantly increases root system root-hair zone length and root gross density, and then indirectly expands the contact area of root nodule bacterium and host's root system.
4. new opplication according to claim 1, is characterized in that, compared with Wild-type soy, the genetically engineered soybean of overexpression GmEXPB2 significantly increases and infects line quantity, accelerates the formation of the former base of root nodule.
5. new opplication according to claim 1, is characterized in that, compared with Wild-type soy, the genetically engineered soybean of overexpression GmEXPB2 significantly increases root nodule numbers, weight and nodule nitrogenase activities.
6. new opplication according to claim 1, is characterized in that, compared with Wild-type soy, the genetically engineered soybean of overexpression GmEXPB2 significantly promotes plant strain growth, adds nitrogen/phosphorus content and biomass.
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| CN110055271A (en) * | 2019-01-18 | 2019-07-26 | 华南农业大学 | A kind of Symbiotic effectiveness method promoted between rhizobium and plant |
| CN110095558A (en) * | 2019-05-20 | 2019-08-06 | 山西大学 | A kind of method of quick measurement soybean kernel protein content |
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| US20040132617A1 (en) * | 1997-05-02 | 2004-07-08 | The Penn State Research Foundation | Beta-expansins as cell wall loosening agents, compositions thereof and methods of use |
| CN102154322A (en) * | 2011-03-14 | 2011-08-17 | 华南农业大学 | Application of gene GmEXPB2 in improving phosphorus absorption efficiency of soybean plants |
-
2015
- 2015-03-23 CN CN201510129067.XA patent/CN104673829B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040132617A1 (en) * | 1997-05-02 | 2004-07-08 | The Penn State Research Foundation | Beta-expansins as cell wall loosening agents, compositions thereof and methods of use |
| CN102154322A (en) * | 2011-03-14 | 2011-08-17 | 华南农业大学 | Application of gene GmEXPB2 in improving phosphorus absorption efficiency of soybean plants |
Non-Patent Citations (3)
| Title |
|---|
| WENBING GUO等: "A soybean β-expansin gene GmEXPB2 intrinsically involved in root system architecture responses to abiotic stresses", 《THE PLANT JOURNAL》 * |
| XINXIN LI等: "GmEXPB2, a Cell Wall b-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development", 《PLANT PHYSIOLOGY》 * |
| 王树起等: "缺磷胁迫对大豆根瘤生长和结瘤固氮的影响", 《大豆科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110055271A (en) * | 2019-01-18 | 2019-07-26 | 华南农业大学 | A kind of Symbiotic effectiveness method promoted between rhizobium and plant |
| CN110055271B (en) * | 2019-01-18 | 2022-09-27 | 华南农业大学 | Method for promoting symbiotic effect between rhizobia and plants |
| CN110095558A (en) * | 2019-05-20 | 2019-08-06 | 山西大学 | A kind of method of quick measurement soybean kernel protein content |
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| Publication number | Publication date |
|---|---|
| CN104673829B (en) | 2017-08-08 |
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